Investigations on the dynamic structural and conformational change of nucleic acid may reveal its interaction with environment at molecular level. This invention provides a powerful method, unique apparatus and convenient kits for the investigation of the structure and conformation of these nucleic acid molecules in real time. The methods in this invention transform the nucleic acid molecule into its own fluorescent probe and force it to reveal its interactions with other nucleic acids, proteins and biomolecules or complexes in vitro and in vivo.
Generally, nucleic acid molecules under study are chemically synthesized oligo(deoxy)nucleotides or oligonucleotides. In this invention, oligonucleotides (ON) is used for the general discussions concerning nucleic acid study.
1) Fluorescent Base Analogues as Nucleic Acid Molecule's Intrinsic Probe
Since the ON molecules under study have the same nucleotides with the nucleic acids molecules in the biological matrix, the former approaches to the detection of these molecules have to use extrinsic probes. Current approaches for the detection of nucleic acid molecules' structure and interactions typically rely on fluorescence resonance energy transfer (FRET) between a fluorophore and a quencher molecule or a second fluorophore (e.g., a fluorescence resonance energy transfer system). Two fluorophores are attached to different nucleic acids. FRET occurs when the two fluorophores are brought into proximity under exciting radiation. The direct correlation of measurable FRET efficiency with the distance between the two chromophores has made FRET one of the most extensively used methods to investigate molecular interactions. A reversed approach is called molecular beacon. It utilizes nucleic acid probes bearing a fluorophore and a quencher molecule. The probes were self-complementary and adopted a hairpin conformation in solution. The hairpin juxtaposed the fluorophore and the quencher thereby reducing or eliminating fluorescence of the fluorophore. When the probes hybridized to a target nucleic acid, they unfold into linear conformation, thus separate the fluorophore from the quencher molecule and reveal a detectable fluorescent signal.
Both of these approaches required an extrinsic fluorescent probe and a second fluorophore or a quencher. Most extrinsic fluorescent probes and their binding moieties are relatively large. The presence of bulky fluorescent labels and associated linkers not only alter the mobility of the nucleic acid but also change the interaction of the nucleic acid so labeled with other molecules either through chemical interactions or through steric hindrance. Therefore, the use of these extrinsic markers in the study of nucleic acid molecule may not reflect its real behavior.
This invention utilizes fluorescent base analogue substitutions serving as intrinsic probes. Representative fluorescent base analogues are presented in FIG. 1. A major feature of these base analogues is that they are designed as structural analogs to the natural bases to minimize the perturbation of the formation of helix and normal interaction with enzymes other biomolecules. They exhibit much higher fluorescence quantum yields than the natural bases. Like in natural nucleosides, they are incorporated into the oligonucleotide through a (deoxy)ribose linkage. Some of them have been produced as phosphoramidites, therefore they can be site-specifically incorporated into DNA using automated DNA synthesis. Their fluorescence intensities are very sensitive to the conformational change and binding situations, therefore are suitable as probes in characterization of nucleic acid molecule's behavior.
In this invention, one or several fluorescent base analogue(s) is (are) substituted into the nucleic acid molecule to replace the corresponding biological base(s). The substituting fluorescent base in the nucleic acid sequence serves as intrinsic fluorescent probe to facilitate comprehensive characterization of nucleic acid molecule' behavior under the influence of the medium or matrix. The methods generally substitute one or more fluorescent base analogue into a nucleic acid (e.g., an oligonucleotide) to replace the corresponding normal base(s) and test its fluorescent properties under the target condition. The substitution of the fluorescent base analogues within the nucleic acid sequence renders them exquisitely sensitive to changes in conformation and integrality as the nucleic acid meets and reacts with other molecules. Subtle structural and conformational changes, like bending, annealing, binding, digestion or cleavage of these fluorescent base analogues-containing nucleic acid molecules can be detected by monitoring changes in fluorescence properties, such as the fluorescence intensity, anisotropy, lifetimes, spectral shifts, and energy transfer characteristics.
2) Fluorescence Properties of Fluorescent Base Analogues
Fluorescence spectroscopy has the advantage of high detection capability, high selectivity and high sensitivity to intermolecular interactions. The following fluorescence properties may be used to characterize the nucleic acid molecule:                Excitation wavelength        Excitation absorbance        Emission wavelength        Fluorescence intensity        Energy transfer efficiency        Fluorescence Lifetime        Anisotropy        
Three new findings of the properties of the incorporated fluorescent base analogues serve as the theoretical foundation of this invention:                1. The modulation of fluorescence properties of the incorporated fluorescent base analogue may be directly correlated to the structure change and interaction feature of the nucleic acid molecule so modified.        2. Sequence dependent energy transfer inside nucleic acid molecule is generally feasible between the normal bases and well designed fluorescent base analogues;        3. Juxtapositioned identical fluorescent bases in the nucleic acid sequence may form dimmer and have pronounced excimer fluorescent emissions.        
Fluorescence activity of a fluorophor may be quenched as a result of interaction of either the ground or excited state of the other species in solution. Interaction between the quencher and the excited fluorophore is called dynamic or collisional quenching; relatively stable complex formed between the quencher and the potentially fluorescent species in the ground state is called static quenching. Although both of these quenching mechanisms influence the fluorescence emission of the base analogues, the static quenching is more pronounced for fluorescent bases incorporated into the nucleic acid molecules. Staked in the sequence, base analogues' fluorescence may be substantially quenched up to 90%. It has been discovered that the disturbance of the stacking position of the fluorescent bases may reduce or eliminate the quenching and recover the quenched fluorescence in some level depending on the interaction with other molecules. For example, if the substituted base analogue is flipped out by enzyme interaction into open environment, the fluorescent may be fully recovered; if repairing enzymes excise or replace the substituted base analogues, their fluorescence activity will be restored to their free monomer level. This recovery may result in more than 10-fold increase of fluorescence from the quenched stacking configuration.
It is further found that, as a general phenomenon, the pronounced fluorescence of the base analogues in aqueous environment will be quenched when it is incorporated into a more hydrophobic condition, such as hybridization into double helix, bound as a substrate into an enzyme, or enveloped into cellular organelles. The fluorescence of single strand nucleic acid molecule, or even double strand nucleic acid molecule may be further reduced by these kinds of hydrophobic bindings. Thus, the change of fluorescence properties of the nucleic acid molecule with substituted fluorescent base analogue may characterize the interaction of the nucleic acid molecule in a qualitative manner.
Energy migration along the DNA bases has attributed to the DNA lesions far from the absorbing site. It has been discovered that, excited normal bases may transfer their energy to the adjacent fluorescent base analogues, and excite them for fluorescence emission. This phenomenon is different from the fluorescence resonance energy transfer (FRET). FRET is a comparatively long-range phenomenon. In FRET, the excitation of the donor results the fluorescence emission, which has to be in the excitation range of the acceptor. If nearby acceptor is in the parallel transition dipole orientation, it will become excited and subsequently undergoes the same physical and chemical processes as if excited directly. However, Energy transfer in DNA or RNA is believed to be electron exchange or charge transfer. It doesn't need donor's fluorescence emission to excite the acceptor like in FRET, but it requires some overlap of the electron orbitals of the excited donor and the acceptor. Instead of the relatively long-range interaction in FRET (10-100 Angstroms), energy transfer in DNA/RNA needs much closer proximity and more precise orientation. It is a discovery of this invention that, DNA/RNA's adjacent base distance (3.4-3.6 Angstroms) and base stacking arrangement make this kind of intrasequence energy transfer generally available for structurally similar base analogues.
This invention utilizes the intrasequence energy transfer from normal bases to fluorescent base analogues to serve as indirect excitation of the fluorescent analogues. This method ties the fluorescent emission of the base analogues to the normal bases, thus it is more sensitive to the structural and conformational change of the whole nucleic acid molecule.
The aggregation of an excited-state molecule with a ground state molecule produces an excited state complex (“exciplex”). A special case of exciplexes occur if the excited-state and ground-state molecules are of the same kind, which is then called an excited state dimer (“excimer”). This species is a charge-transfer complex that is held together by favorable orbital interactions as well as by Coulombic binding forces. Exciplexes are distinct intermediates in their own right and possess unique properties. For instance, their fluorescence excitation and emission is almost always at longer wavelengths (lower energy level) than that of the excited state. This short-range phenomenon is only significant when the electron orbitals of donor and acceptor overlap. It is most efficient when the exciplexes formed by the same kind of molecules. Therefore, excimer is the most favored case in exciplex phenomena.
In general, the close proximity and precise spatial stacking of nucleotide in sequence facilitates excimer formation. If identical base analogues are put into nucleic acid molecule at adjacent positions, intramolecular base excimer may forms from the excitation of ground state dimerization, or from the relaxation of the excited individual bases. Although the fluorescence of excimer is weaker, its red-shifted wavelength and sensitivity may be used for nucleic acid molecule conformation detection. In addition to the binding sensitivity common for other labeling arrangement, the excimer formation delicately depends on the stacking of the adjacent bases. If the base stacking were disturbed, the fluorescence of the excimer will decrease or disappear.
This invention makes use of excimer fluorescent emission to facilitate another way to tie the fluorescence signal of the base analogues with the whole nucleic acid molecules. It is especially suitable for structural change detection of double strand oligonucleotides. Excimer formation in nucleic acid also generates a fluorescent signal in the further red shift of the cell auto-fluorescence range. The longer wavelength emission by the excimer further distinguishes the signal from background fluorescence of the biological matrix of the nucleic acid molecules, thus makes the measurement more reliable.
In summary, this invention provides a novel method, corresponding apparatus and kits to make quantitative and qualitative investigation of nucleic acid molecules' structure and interaction. By using fluorescent base analogues as intrinsic probes, this invention ties the fluorescent base analogues within the nucleic acid sequence and transforms the nucleic acid molecule into a fluorescent probe of itself to demonstrate its behavior in real time.